The exocrine pancreas is responsible for important fluid and electrolyte secretions, but efforts to understand these processes at a cellular level have been hampered by the complex anatomy and cellular heterogeneity of this organ. The objective of this proposal is to dissociate the pancreas into its component cell types and develop cultures of pancreatic acinar and ductal cells on floating collagen gels and then to use these monolayers as reconstituted epithelia in an investigation of pancreatic fluid and electrolyte secretion. Pancreatic cells will be isolated from mice and various procedures for purification of acinar and ductal elements evaluated. Both unfractionated and purified cells will be placed in culture on collagen gels, a method which has proven superior for maintaining differentiated structure and secretory ability of other exocrine gland cells (e.g. mammary epithelium, parotid gland). Various hormonal regimens, extracellular matrix requirements and culture conditions will be tested for their effects on differentiated structure and function by electron microscopic, biochemical and electrophysiological means. Production of pancreatic monolayers will allow employment of radioisotope fluxes (36Cl-, 22Na+, 45Ca++, 88Rb+) and Ussing-chamber electrophysiological measurements (transepithelial potential difference, resistance, and short-circuit current) to analyze the movement of the ions Na+, K+, Cl-, Ca2+, and HCO3- which are involved in the formation of pancreatic juice. The use of short-circuit current techniques, along with ion substitution experiments, and the use of pharmacological inhibitors of ion transport, such as ouabain, amiloride and bumetanide, will provide detailed information about pancreatic ion transport mechanisms. The use of monolayers composed of purified cell types will allow assignment of ion transport characteristics of individual cell types from this heterogenous and anatomically complex organ. The effects of secretagogues known to regulate pancreatic secretion in vivo, including cholecystokinin, acetylcholine and secretin, as well as those of the pharmacological secretagogues dibutyryl cAMP and the calcium ionophore A23187, will be studied. This information will be used to develop a cellular model of normal pancreatic fluid and electrolyte secretion which will serve for future basic research and for comparison with various disease states.